Instrument: Illumina Genome Analyzer
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Worm_embryo_extraction_v5. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) + protease inhibitors (Calbiochem Cat# 539131)+phosphatase inhibitors(Calbiochem cat#524624). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC and taking the supernatant. Protein concentration was determined by Bradford Assay and extracts were aliquoted at stored at -80C. For a detailed protocol see http://www.modencode.org/. Worm_chromatin_immunoprecipitation_v5. 2mg extract + 1%sarkosyl was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 50 ul of IgG dynabeads (Dyna), and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac. DNA for Library Prep is incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends. It is then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3? ends (a single A-overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments is ligated with single-end ?Homebrew? adaptors which contain an index sequence within (Corbin Jones? lab). After ligation, the samples are purified TWICE using SPRI beads, allowing for a size selection step getting rid of excess adaptors. Samples are then amplified by PCR with single end primers. Depending on the original fragmentation of the DNA, samples can either be purified by SPRI or by separation and purification from an agarose gel. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.